The tremendous impact of influenza virus infections on the public health is widely recognized. Control of influenza has relied primarily on the use of inactivated influenza vaccines. More current approaches, however, have moved towards the use of live attenuated vaccine. Kilbourne, E. D. “Influenza” (Plenum Publishing Corp. New York), p. 291-332 (1987). The most promising efforts in the development of an effective live vaccine have centered on adapting the virus to grow at suboptimal temperatures. Maassab, H. F., et al., Vaccine 3:355-369 (1985). Using this approach, cold-adapted attenuated influenza viruses have been developed in both the former Soviet Union and the United States. Alexandrova, G. I., et al., Rev. Roum. Inframicrobil. 2:179-189 (1965); Maassab, H. F. Nature (London) 213: 612-614 (1967).
In particular, cold adaptation (ca) has permitted the A/Ann Arbor/6/60 (H2N2) (A/AA/6/60) virus of the present invention to grow as well at 25° C. as it does at 33° C. Maassab, H. F. Nature (London) 213:612-614 (1967); Maassab, H. F. “Biology of Large RNA Viruses” (Academic Press, New York), p. 542-565 (1970). The ca A/AA/6/60 virus is also temperature-sensitive (ts), a property that impedes replication at higher temperatures in the lungs and thus is highly desirable for live vaccines. Maassab, H. F., “Biology of Large RNA Viruses” (Academic Press, New York), p. 542-565 (1970); Mulder, J., et al., “Influenza” (Wolters-Noordhoff, Amsterdam), 1-6:78-80 (1972). Single-gene studies of this cold-adapted virus in a background of A/Korea/1/82 (H3N2) have identified the genes responsible for the ca and ts phenotypes and for attenuation in that gene constellation. Snyder, M. H., et al., J. Virol. 62(2):488-495 (1988).
Live attenuated vaccines are produced by reassorting the six internal genes of the cold-adapted A/Ann Arbor/6/60 influenza virus with the two surface genes of the currently circulating wild type (wt) virus, thereby producing a reassortant strain. Maassab, H. F. “Negative Strand Viruses” (Academic Press, New York), p. 755-763 (1975); Davenport, F. M., et al., J. Infect. Dis. 136:17-25 (1977). Vaccines prepared from ca A/AA/6/60 have proven both non-reactogenic and non-transmissible in preliminary field trials at six different medical centers involving over 20,000 people. Couch, R. B., et al., “Options for the Control of Influenza” (Alan R. Liss, New York), p. 223-241 (1986); Wright, P. F., et al., “Options for the Control of Influenza” (Alan R. Liss, New York), p. 243-253 (1986). These vaccines also provide higher IgA levels than the killed vaccines and afford longer-lasting protection in children. Murphy, B. R., et al., Infect. Immun. 36(3):1102-1108 (1982); Johnson, P. R., et al., J. Infect. Dis. 154(1):121-127 (1986). Currently, the ca A/AA/6/60 7PI (plaque-purified seven times) master strain preparation is under development for use as a live vaccine in children and other live virus vaccines are being developed using the live ca influenza vaccine as a model.
Cold-adapted reassortant vaccines have thus been shown to have the proper level of attenuation, immunogenicity, and non-transmissibility combined with proven genetic stability and are produced in acceptable tissue culture substrates. In general, live cold-adapted reassortant vaccines offer several advantages over the existing inactivated vaccine. These include the possible use of a single dose, and administration by the natural route of infection, i.e. intranasally. In addition, ca vaccines stimulate a wide range of antibody responses, and result in induction of both local and humoral immunity. Furthermore, these vaccines are cost-effective and can be rapidly produced and updated in the event of antigenic changes. In addition, laboratory guidelines are available for the assessment of virulence (reactogenicity in ferrets) and attenuation can be reproducibly achieved. Moreover, the presence of two phenotypic markers (the temperature-sensitive and cold-adapted phenotypes) allows for the evaluation of virulence and monitoring of the vaccine in the field.
However, despite the above-described advantages, until now virtually nothing has been known about the molecular basis of cold adaptation. Published information indicates that cold adaptation has produced one or more mutations in each of the genes encoding the internal proteins of the A/AA/6/60 master strain. Cox, N. J., et al., “Genetic Variation Among Influenza Viruses” (Academic Press), p. 639-652 (1981). However, all of the work has been done on viruses passaged 28 to 32 times in eggs in parallel with the virus passaged in primary chick kidney cells during cold adaptation. Cox, N. J., et al., Virol. 167:554-567 (1988). Studies, however, have shown a gradual buildup of mutations in the RNA1 of sequential 35° C. egg passages 2 through 28 of wild type viruses, and recent findings have shown the influence of host cell variation on influenza viruses passaged in chicken eggs. Katz, J. M., et al., Virol. 156:386-395 (1987). Thus, the mutations leading to cold adaptation and attenuation have heretofore been unknown.
It would thus be desirable to isolate and provide the wild type A/Ann Arbor/6/60 progenitor virus and determine the accurate nucleic acid sequence of its genome. It would further be desirable to identify the mutations leading to cold adaptation, thus accurately characterizing the nucleic acid sequence of the ca master strain. It would also be desirable to produce and provide cold-adapted influenza strains through reassortment with currently circulating wild type strains. It would also be desirable to produce and use a cold-adapted influenza vaccine to prevent and/or treat influenza.